Probiotic bacteria (referred to here in some cases as probiotics) are live bacterial microbes that beneficially influence the health and nutrition of individuals by promoting a healthier microflora in the host's intestine. These microflora are dependent on substances fed to them from the diet of the host organism. Probiotics typically colonize in the large intestine and can serve either or both of at least two major roles: they can supplement the natural flora of the gastrointestinal tract with additional bacteria, and they can be effective in treating a number of health conditions, including, but not limited to (1) alleviation of intestinal disorders (e.g., constipation and diarrhea caused by an infection by pathogenic organisms, antibiotics, chemotherapy, etc.); (2) stimulation and modulation of the immune system; (3) anti-tumoral effects resulting from inactivation or inhibition of carcinogenic compounds present in the gastrointestinal tract by reduction of intestinal bacterial enzymatic activities (e.g., O-glucuronidase, azoreductase, nitroreductase, etc.); (4) reduced production of toxic final products (e.g., ammonia, phenols, other protein metabolites known to influence hepatic cirrhosis, etc.); (5) reduction of serum cholesterol and arterial pressure; (6) maintenance of mucosal integrity; (7) alleviation of lactose intolerance symptoms; and/or (8) prevention of vaginitis.
Potential mechanisms of anti-pathogenic effects of probiotic bacteria are through decreasing the luminal pH by the production of short chain fatty acids such as acetic acid, lactic acid or propionic acid, rendering vital nutrients unavailable to pathogens, altering the redox potential of the environment, producing hydrogen peroxide or producing bacteriocins or other inhibitory substances (Kailasapathy and Chin, 2000). In recent years, the specific live microbial food ingredients and their effects on human health have been studied both within food matrices and as single or mixed culture preparations. Due to their perceived health benefits, probiotic bacteria have been increasingly included in fermented dairy products. Probiotics have been incorporated into fermented milks, yoghurts, soft, semi-hard and hard cheese, ice cream, and frozen fermented dairy desserts. Some of the most common types of probiotic bacteria include Lactobacillus and Bifidobacteria (Axelsson, 1993; Holzapfel et al., 2001).
The ability of probiotic microorganisms to survive and multiply in the host strongly influences their probiotic benefits. The bacteria should be metabolically stable and active in the product, survive passage through the upper digestive tract in large numbers and have beneficial effects when in the intestine of the host (Gilliland, 1989). The typical standard for any food sold with health claims from the addition of probiotics is that it contains at least 109-1010 colony forming units (CFU) of viable probiotic bacteria per serving. Probiotics are sensitive to various environmental conditions and typically lack the ability to survive for long periods of time in “high acid” foods and beverage products (e.g., fresh citrus fruits, citrus fruit juices, foods containing citrus fruit juices, tomato sauce, etc.). For example, in fruit juice beverage products probiotics are sensitive to numerous environmental conditions, including, e.g., low pH, high acid content, high water activity, heat, air, light, and the inherent presence of polyphenols found in fruit juices, or other environmental influences. Thus, the viability (measured in colony forming units or CFU), and therefore the efficacy, in comestibles supplemented with probiotics and in the gastrointestinal tract can be substantially reduced.
If an edible composition has a pH of less than 7 it is considered acidic. The acids present in an edible composition (e.g., a food or beverage product) contribute to the pH level. The more acid present, the lower the pH is likely to be. High-acid edible compositions are generally considered to have a natural pH of 4.6 or below. For example, one of the dominant nutrients in citrus fruit is acid, e.g., ascorbic acid (Vitamin C), and the pH level of orange juice is around 3.8. Acidic environments are known to denature vital proteins necessary for the growth of bacterial organisms. Consequently, the organisms die in an acidic environment. Many desirable probiotics grow best at pH values around 7.0. The terms “acid content” and “degree of acidity” can be distinguished. The acid content is a measure of how much acid is present per unit volume of the edible composition. The degree of acidity is the actual pH value of the food or beverage. A high acid content gives a lower pH value, whereas a low acid content results in a higher pH value.
Heat (e.g., in the form of pasteurization) is routinely used to kill microbes that may be present in foods. In general, the cooler a product can be maintained, the greater the probiotic survival. Sunlight or artificial light can also kill at least some probiotics. Certain wavelengths of UV light are especially harmful. Due to probiotic sensitivity, environmental influences like high temperatures, high oxygen levels, moisture and direct light may result in beverages containing these organisms having a short shelf life. The result is a product with an inadequate shelf life, that is, a product whose decreased probiotic cell count determines the end of the product's shelf life, leading to higher costs and increased waste.
Encapsulation techniques, such as microencapsulation, have been investigated for use to enhance processing, storage and digestive stability of sensitive materials, such as probiotic bacteria, allowing stabilization and temporal and targeted release of ingredients. Microencapsulation has been defined as a technology of packaging solids, liquids or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under the influences of specific conditions (Anal and Stevens, 2005; Anal at al., 2006). Microencapsulation has been used to enhance processing, storage and digestive stability of sensitive materials, such as probiotic bacteria. This technology allows materials to be coated or entrapped in a matrix creating a barrier to the surrounding environment, which is subsequently degraded to release the core material. The composition of microcapsules may be manipulated to improve stability and allow degradation under specific conditions (Anal and Singh, 2007). The goal of microencapsulation of probiotic bacteria is thus to prevent damage during processing and storage and from degradation by gastric acid, proteolytic enzymes and bile salts before targeted release in the colon.
To date, the research on encapsulation of probiotics has mainly focused on maintaining viability of probiotic bacterial cells at low pH and high bile concentrations, as well as during spray drying, freeze drying and storage. Much research has focused on microencapsulation technologies and the manipulation of encapsulation parameters, such as coating material types and their concentrations and the use of multiple coating layers. A few attempts have been made to improve the viability of probiotics at high temperatures by adding thermoprotectants, however the viability has been found to be negligible with many strains. Consequently, there appear to be no commercial probiotic products available that are stable at high temperatures. Moreover, prior encapsulation methods have required employment of water-in-oil or oil-in-water emulsions, multiple reaction steps, multiple encapsulation coatings or shells, or combinations thereof.
Consumers demonstrate continued interest in comestible products such as ready-to-drink (RTD) beverages or foods fortified with ingredients believed to provide health benefits. It would be desirable to provide probiotics or other nutrients in a stable form for use in comestible products, so that the ingredients can withstand certain process conditions related to processing (e.g., mixing, homogenizing, pasteurizing, etc.) of the comestible, yet would be available as a nutrient within the gastrointestinal tract, once the food or beverage is consumed by an individual.
Various documents including, for example, publications and patents, are recited throughout this disclosure. All such documents are hereby incorporated by reference. The citation of any given document is not to be construed as an admission that it is prior art with respect to the present invention. To the extent that any meaning or definition of a term in this written document conflicts with any meaning or definition of the term in a document incorporated by reference, the meaning or definition implied or assigned to the term in this written document shall govern.